Pharmaceutical composition for preventing or treating metabolic diseases, comprising bacteroides acidifaciens as active ingredient

ABSTRACT

The present disclosure relates to a composition for preventing or treating metabolic diseases, in which the composition includes  Bacteroides acidifaciens  as an active ingredient. In addition, the present disclosure relates to a composition for oxidizing fat or inhibiting DPP-4, in which the composition includes  Bacteroides acidifaciens  as an active ingredient. In addition, the present disclosure relates to a transformant expressing a lean phenotype, in which an Atg7 gene is deleted in dendritic cells.

TECHNICAL FIELD

The present disclosure relates to a composition for preventing ortreating metabolic diseases, in which the composition includesBacteroides acidifaciens as an active ingredient.

In addition, the present disclosure also relates to a composition foroxidizing fat or inhibiting DPP-4, in which the composition includesBacteroides acidifaciens as an active ingredient.

BACKGROUND ART

A metabolic disease is a syndrome that appears with the risk factorssuch as obesity, diabetes, hypertension and arteriosclerosis caused byexcessive nutrition accumulation in the body and lack of exercise. Inrecent years, it has been formally named the metabolic syndrome orinsulin resistance syndrome through the Adult Treatment Program PanelIII established by the World Health Organization and the National Heart,Lung, and Blood Institute of the National Institutes of Health. Inaddition, according to the ATP of the USA National Cholesterol EducationProgram (NCEP) announced in 2001, it is judged to be a metabolic diseaseif a patient showing at least three of the five risk factors ofabdominal obesity with a waist circumference of 40 inches (102 cm) orlonger for men and 35 inches (88 cm) or longer for women, triglyceridesof 150 mg/DL or higher, HDL cholesterol of 40 mg/dL or less for men and50 mg/dL or less for women, blood pressure of 130/85 mmHg or higher, andfasting glucose of 110 mg/dL or higher. In case of Asians, it has beensomewhat adjusted that the waist circumference of 90 cm or longer formen and 80 cm or longer for women is considered as abdominal obesity.When these rules are applied, there is a recent study showing thatapproximately 25% of Koreans among the entire population shows metabolicsyndrome symptoms.

On the other hand, mucosal immune tissues refer to tissues covered withmucous membranes ranging from respiratory organs, genital organs anddigestive organs, and these tissues are directly connected to theexternal environment and are easily exposed to foreign antigens andpathogens. In the mucosal tissues of a human body, variousmicroorganisms of nearly 100 trillion such as bacteria, fungi, protozoa,etc. produce clustering, and coexist with them.

In comparison of systemic immune tissues, mucosal immune tissues have animmunological tolerance mechanism to coexist with various symbioticmicroorganisms, and at the same time, it has a system that can causerapid and powerful immune response for the primary defense againstpathogenic microorganisms.

Intestinal microorganisms are known to affect human health and diseasesby participating in the maintenance of intestinal homeostasis andmetabolic regulation through many mechanisms. Intestinal microorganismsferment undigested polysaccharides to prepare short chain fatty acidsand supply the energy source of intestinal epithelial cells. Intestinalmicroflora of a human being may be broadly divided into four phyla ofgram-negative bacteria, Bacteroidetes and Proteobacteira, andgram-positive bacteria, Firmicutes and Actinobacteria.

In particular, obesity is one of the health risk factors associated withdiseases such as cardiovascular disease, diabetes and osteoporosis.Recently, many research results have been published showing that obesityis deeply related to changes and diversity of intestinal microflora. Incomparison of the intestinal microorganisms of an obese mouse (ob/obmouse) with those of a normal body weight mouse, it is known thatFirmicutes phylum is increased and Bacteroides phylum is decreased.Similarly, it has been reported that when low-carbohydrate or low-fatmeals are served to obese humans, Bacteroidetes phylum is increased, andin a study of twins, the diversity is decreased and Bacteroidetes phylumis decreased on analysis of intestinal microorganisms of obese humans.

As a result of analyzing intestinal microorganism genetic makeups inlean and obese humans, it was confirmed that there are significantdifferences in the type and amount of intestinal microbial species. Inother words, the result has been reported that obese patients who do nothave abundant intestinal microorganisms show symptoms such as adiposity,insulin resistance, dyslipidemia, and inflammatory reaction, and gainbody weight more easily than obese humans with abundant intestinalmicroorganisms.

The interaction between intestinal microorganisms and hosts play animportant role in the pathogenesis of obesity and metabolic syndrome.There is a high possibility of preventing/treating obesity byinvestigating and isolating/identifying the role of microorganisms thatare symbiotic in the intestine and causing changes in these interactionsusing the concept of probiotics.

However, it has not yet been proven that certain intestinalmicroorganisms are directly involved in lipid metabolism and can affectbody weight and fat mass.

DISCLOSURE Technical Problem

Accordingly, the present inventors have established a transgenic mouseshowing a lean phenotype, specified specifically increased intestinalmicroorganisms in the mouse, and confirmed that the correspondingmicroorganisms can be effectively involved in internal metabolism,particularly lipid metabolism, and completed the present disclosure.

Accordingly, one aspect of the present disclosure is to provide acomposition for preventing or treating metabolic diseases, in which thecomposition includes the effective microorganisms.

Another aspect is to provide a method for preventing or treatingmetabolic diseases, in which the method includes administering theeffective microorganisms to a subject in need of prevention or treatmentof metabolic diseases. The method may further include, prior toadministering, identifying a subject in need of prevention or treatmentof metabolic diseases (e.g., identifying whether the subject foradministration is suffering from or run a risk of developing a metabolicdisease).

Another aspect is to provide a use of the above effective microorganismsto be used for the preparation of a pharmaceutical composition forpreventing or treating metabolic diseases, or for preventing or treatingmetabolic diseases.

In addition, one aspect of the present disclosure relates to acomposition for oxidizing fat or inhibiting DPP-4 or a composition forpreventing or treating diseases associated therewith, in which thecomposition includes the above effective microorganisms.

In addition, another aspect of the present disclosure relates to amethod for inhibiting DPP-4 or promoting fat oxidation or a method forpreventing or treating diseases associated therewith, in which themethod includes administering the effective microorganisms to a subjectin need of DPP-4 inhibition. The method may further include, prior toadministering, identifying a subject in need of DPP-4 inhibition or fatoxidization, or a subject in need of prevention or treatment of thediseases associated therewith.

Another aspect relates to a use of the above effective microorganisms tobe used for the DPP-4 inhibition or fat oxidization or for preventing ortreating the diseases associated therewith, or a use of the aboveeffective microorganisms to be used for the preparation of apharmaceutical composition for inhibiting DPP-4 or oxidizing fat or forpreventing or treating the diseases associated therewith.

Another aspect of the present disclosure relates to a transformantexpressing a lean phenotype, in which an Atg7 gene is deleted indendritic cells.

Another aspect relates to a method for preparing a transformantexhibiting a lean phenotype, in which the method includes deleting anAtg7 gene in dendritic cells.

In addition, another aspect of the present disclosure relates to apharmaceutical composition for preventing or treating metabolicdiseases, in which the pharmaceutical composition includes Atg7, and anexpression inhibitor or an activity inhibitor of a gene coding the sameas active ingredients.

Technical Solution

One aspect of the present disclosure provides a pharmaceuticalcomposition for preventing or treating metabolic diseases, in which thepharmaceutical composition includes Bacteroides acidifaciens as anactive ingredient. Another aspect provides a method for preventing ortreating metabolic diseases, in which the method includes administeringBacteroides acidifaciens to a subject in need of prevention or treatmentof metabolic diseases. The method may further include, prior toadministering, identifying a subject in need of prevention or treatmentof metabolic diseases. Another aspect is to provide a use of Bacteroidesacidifaciens to be used for the preparation of a pharmaceuticalcomposition for preventing or treating metabolic diseases or forpreventing or treating metabolic diseases.

Hereinafter, the present disclosure will be described in detail.

In a specific example, the present inventors confirmed that when an Atg7gene was deleted in dendritic cells, a lean phenotype was expressed inthe mouse, and then the intestinal microorganisms were compared with acontrol group, and confirmed that Bacteroides acidifaciens was presentat a high level in an Atg7^(ΔCD11c) mouse expressing a lean phenotype.In addition, it has been confirmed that Bacteroides acidifaciens reducesthe body weight and body fat of a mouse, reduces blood glucose levels,and increases blood insulin production.

In addition, Bacteroides acidifaciens (BA) according to the presentdisclosure causes fat oxidation activity through a bile acid-TGR5-PPARαaxis in adipose tissues, resulting in high energy consumption. At thesame time, BA activates visceral DPP-4, followed by the increase inGLP-1, thereby contributing to glucose homeostasis. Bile acids, cholateand taurine contributed to GLP-1 activity through a TGR5 receptor andimproved insulin sensitivity.

Accordingly, Bacteroides acidifaciens of the present disclosure may beused as a pharmaceutical composition for preventing or treatingmetabolic diseases.

The above Bacteroides acidifaciens is a concept including not onlymicrobial cells themselves but also cultures of the bacteria, forexample, cultures containing microbial cells or cultures excludingmicrobial cells, dried materials, fragments, fractions of the cultures,and the like.

The term “metabolic disorder” used in the present specification refersto a disease selected from the group consisting of obesity, diabetes,diabetic complication, fatty liver, hypertension, peripheral vasculardisease, dyslipidemia, insulin resistance, cardiovascular disease,arteriosclerosis, metabolic syndrome, hyperglycemia, hyperlipidemia,carbohydrate metabolic abnormality, and the like.

Obesity is an example of the metabolic disease. The term “obesity” inthe present disclosure is a disease defined as an increase in the numberof adipocytes and an expansion of cell volume, and may be a major causeof metabolic diseases such as diabetes, hypertriglyceridemia,hypertension, cardiovascular disease, blood coagulation defect kidneydisease, eye disease, and foot infection, etc.

In addition, as another example of metabolic diseases, “diabetes” is adisease in which insulin is deficient or insulin sensitivity is reduced,resulting in an abnormality in carbohydrate metabolism, and is dividedinto type 1 diabetes which results from a condition that the pancreasproduces little insulin (insulin-dependent diabetes mellitus: IDDS) andtype 2 diabetes which begins with a rejection of tissue to insulin(non-insulin-dependent mellitus: NIDDM).

More than 90% of all diabetes is type 2 diabetes. Type 2 diabetes isknown to be closed associated with heredity and obesity, especiallyabdominal obesity (a state that a ratio of waist circumference: a hipcircumference is 85:100 or more). Accordingly, in the presentdisclosure, diabetes preferably means type 2 diabetes. The metabolicdiseases may include complications of diabetes. Acute complications ofdiabetes (hypoglycemia, ketoacidosis or nonketotic hyperosmolar coma)can occur when diabetes is not adequately controlled. Prolonged seriouscomplications include cardiovascular disease (double risk), chronicrenal failure, retinal damage that can lead to blindness, many types ofnerve damage, and microvascular damage, which cause erectile dysfunctionand poor healing. Slow healing of wounds (especially, feet) can causegangrene, which can lead to amputation.

In the present specification, the term “fatty liver” refers to aphenomenon in which neutral fat, which is not present in normal cells,is shown to be abnormally deposited in liver cells.

In the present specification, hypertension refers to a state in whichthe blood pressure of arteries is chronically high and an adult over 18years of age has a systolic blood pressure of 140 mmHg or higher, or adiastolic blood pressure of 90 mmHg or higher, and may be caused byobesity.

The term “peripheral vascular disease (PVD)” refers to damage,dysfunction, and the like of peripheral artery and venous.

The dyslipidemia of the present disclosure means a combination of lowhigh-density lipoprotein cholesterol (HDLc), high triglycerideconcentration and slightly high or normal low-density lipoproteincholesterol (LDLc) concentration.

In addition, the insulin resistance of the present disclosure means ametabolic state in which insulin action, which is the most importantbiological hormone for controlling total energy metabolism such ascarbohydrate, lipid and protein, at a physiological insulinconcentration, is declined than normal.

The arteriosclerosis of the present disclosure refers to a state thatfatty substances (plaque) containing cholesterol, phospholipid, calcium,etc. are accumulated in the endangium, and arteries become hardened,lose elasticity and become narrow, thereby causing obstruction to bloodsupply and causing arteriorrhexis, artery dissection, etc. due to highpressure.

The hyperglycemia of the present disclosure refers to a state in whichthe blood sugar level rises abnormally, and may be due to an abnormalityin insulin production or an insulin dysfunction.

The hyperlipidemia of the present disclosure in a condition in whichlipid components such as blood cholesterol increase, blood does not flowsmoothly, the lipid components are adhered to artery walls, resulting ina chronic inflammatory response, and the narrowing of the wall of anartery causes atherosclerosis that leads to hardening of blood vessels.In the long term, thrombosis produced therefrom causes cardiacinfarction, stroke or cerebral infarction, etc. by occluding heartcoronary arteries and cerebral blood vessels.

Hyperinsulinemia means a state in which blood insulin levels are higherthan normal, and there are organic hyperinsulinemia and functionalhyperinsulinemia. The organic hyperinsulinemia is caused byproliferation (adenoma, hypertrophy) of Langerhans islet, resulting inexcessive secretion of insulin from the pancreas, and becoming inspontaneous hypoglycemia. Functional hyperinsulinemia is caused byautonomic nervous system and dysfunction of the digestive system withoutLangerhanssoma adenoma. It means that the insulin level of postprandialdiet is increased mainly by the cause of stroke and hepatitis aftergastrectomy, and hypoglycemia occurs 2 to 4 hours after the meal.

The metabolic disorder of carbohydrate according to the presentdisclosure is a metabolic disease caused by a problem in the process ofglucose biosynthesis for producing glucose from pyruvic acid or aproblem in TCA circuit and oxidative phosphorylation process forproducing carbon dioxide and water from pyruvic acid. Glycogen storagedisease type 1, a fructose-1,6-bisphosphatase deficiency, a pyruvatedehydrogenase complex deficiency, a pyruvate carboxylase deficiency, aglycogenosis, a galactosemia, and the like are the examples thereof, butare not limited thereto.

In addition, as an example of a metabolic disease, there is themetabolic syndrome. The metabolic syndrome is characterized by a groupof metabolic risk factors including the following to those skilled inthe art: abdominal obesity (excess adipose tissue in and around theabdomen); atherosclerotic dyslipidemia (increased blood fat disease—hightriglyceride, low HDL cholesterol and high LDL cholesterol—plaqueformation on arterial walls); increased blood pressure; insulinresistance or glucose hypersensitivity; a prothrombotic state (e.g.,high fibrinogen or PAI-1 (plasminogen activator inhibitor-1) in theblood); and a pro-inflammatory state (e.g., an increase in C-reactiveprotein in the blood). People suffering from metabolic syndrome are atincreased risk for coronary heart disease and other diseases associatedwith plaque buildups on arterial walls (such as stroke and peripheralvascular disease), and type 2 diabetes.

Another example of metabolic diseases includes cardiovascular or cardiacdisorder.

Cardiac disease is a generic term that can be used interchangeably withthe terms such as heart disease and cardiovascular disease. Cardiacdisease in the present specification means every type of diseases thatinhibit the normal functioning of the heart. More specifically, thediseases included in the cardia disease in the present specificationinclude coronary heart disease, cardiomyopathy, cardiovascular disease,ischemic heart disease, heart failure, hypertensive heart disease,inflammatory heart disease and valvular heart disease, but are notlimited thereto.

In addition, one aspect of the present disclosure provides a compositionfor oxidizing fat or a composition for preventing or treating diseasesassociated therewith, in which the composition includes Bacteroidesacidifaciens as an active ingredient. Another aspect provides a methodfor promoting fat oxidization or preventing or treating diseasesassociated therewith, in which the method includes administeringBacteroides acidifaciens to a subject in need of promotion of fatoxidization or prevention or treatment of diseases associated therewith.The method may further include, prior to administering, identifying asubject in need of promoting fat oxidization or prevention or treatmentof diseases associated therewith. Another aspect provides a use ofBacteroides acidifaciens for the production of a pharmaceuticalcomposition for promoting fat oxidation or preventing or treatingdisease associated therewith.

The composition for fat oxidation may be a pharmaceutical compositionfor preventing or treating a physiological or pathological conditioninduced by oxidative stress. In addition, the composition may be apharmaceutical composition for preventing or treating physical or mentalstress. The composition may be a pharmaceutical composition forpreventing or treating one or more diseases selected from the groupconsisting of aging, cancer, multiple atherosclerosis, arthritis,Parkinson's disease, stroke, concussion, Alzheimer's disease, vasculardisorders, hyperlipemia, myocardial infarction and cerebral infarction.

The composition for fat oxidation can be used not only for the metabolicdiseases but also for the treatment of muscle diseases and relateddiseases such as sarcopenia, cachexia, muscle damage, muscular dystrophyand muscle fatigue, the improvement of muscle function and endurance,the improvement of body performance, the increase in endurance ability,the increase in muscle mass, the prevention of muscle loss, the increasein muscle recovery, the reduction of muscle fatigue, the improvement ofenergy balance, the maintenance of muscle performance and/or musclestrength and/or muscle mass and/or muscle function, the improvement ofbody shape, or the improvement of muscle:fat ratio.

In addition, one aspect of the present disclosure provides a compositionfor inhibiting DPP-4 or a composition for hypoglycemia, in which thecomposition includes Bacteroides acidifaciens as an active ingredient.Another aspect provides a method for inhibiting DPP-4 or hypoglycemia,in which the method includes administering Bacteroides acidifaciens to asubject in need of DPP-4 inhibition or hypoglycemia. The method mayfurther include, prior to the administering, identifying a subject inneed of DPP-4 inhibition or hypoglycemia. Another aspect provides a useof Bacteroides acidifaciens for use in the preparation of apharmaceutical composition for inhibiting DPP-4 or hypoglycemia.

Dipeptidylpeptidase-4 (DPP-4) inhibitor is a medicament having a newmechanism for the regulation of blood glucose. Glucagon-like peptide-1(GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) aresecreted in the gastrointestinal tract during food intake. It is calledincretin, secreted in the K and L cells of the intestine, and degradedin the blood by the enzyme DPP-4 in a very short time. DPP-4 inhibitorsincrease the incretin blood level by inhibiting the DPP-4 enzymeresponsible for the incretin degradation. In addition, the use of DPP-4inhibitors has been shown to regulate blood glucose by stimulatinginsulin synthesis and secretion, inhibiting glucagon, and inhibitingglucose synthesis in the liver.

When the composition is prepared as a pharmaceutical composition forpreventing or treating metabolic diseases or a fat oxidation-relateddisease, the composition may include a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers included in thecomposition are commonly used for the preparation, and include lactose,dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calciumphosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup,methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc,magnesium stearate and mineral oil, but are not limited thereto. Thepharmaceutical composition may further include a lubricant, a wettingagent, a sweetening agent, a flavoring agent, an emulsifying agent, asuspending agent, a preservative, etc. in addition to the aboveingredients.

The pharmaceutical composition for preventing or treating metabolicdiseases or fat oxidation-related diseases may be administered orally orparenterally. In the case of parenteral administration, it can beadministered by intravenous injection, subcutaneous injection,intramuscular injection, intraperitoneal injection, endothelialadministration, topical administration, intranasal administration,intrapulmonary administration and intrarectal administration. In thecase of oral administration, protein or peptide is digested and the oralcomposition should be formulated so as to coat the active agent orprotect it from degradation in the stomach. In addition, the compositionmay also be administered by any device capable of transferring theactive material to the target cell.

The subject of administration of the pharmaceutical composition may bean animal such as a mammal such as a human, or a cell, tissue, orculture thereof isolated therefrom.

The appropriate dosage of the pharmaceutical composition for preventingor treating metabolic diseases or fat oxidation-related diseases may beprescribed in various ways depending on factors such as a preparationmethod, an administration method, the age, body weight, gender,pathological condition of a patient, food, administration time,administration route, excretion speed, and responsiveness. The preferreddosage of the composition falls within the range of 100 to 100,000,000(10² to 10⁸) cell/kg on an adult basis. The term “pharmaceuticallyeffective amount” means an amount sufficient to prevent or treat ametabolic disease or a fat oxidation-related disease.

The composition may be prepared in unit dose form by using apharmaceutically acceptable carrier and/or excipient according to amethod which can be easily performed by those skilled in the art, or maybe prepared by inserting it into a multi-dose container. At this time,the formulations may be in the form of solutions, suspensions, syrups oremulsions in oils or aqueous media, or in the form of extract, powders,powders, granules, tablets or capsules, and may additionally containdispersing or stabilizing agents. In addition, the composition may beadministered as an individual therapeutic agent or in combination withanother therapeutic agent, and may be administered sequentially orsimultaneously with a conventional therapeutic agent. It may also beadministered once or additionally, if necessary.

Bacteroides acidifaciens according to the present disclosure may beutilized as a food composition. The food composition of the presentdisclosure may be easily used as a food, for example, main raw materialsand supplementary raw materials of food, food additives, functionalfoods or beverages, which are effective for prevention and improvementof symptoms of metabolic diseases or for fat oxidation.

The term “food” in the present disclosure means a natural product or aprocessed product containing one or more nutrients, preferably a stateof being able to be eaten directly through a certain degree ofprocessing, and as an ordinary meaning, it includes all the food, foodadditives, functional foods and beverages.

Foods for which the composition for prevention or improvement ofmetabolic disease symptoms or for fat oxidization according to thepresent disclosure may include, for example, various foods, beverages,gums, tea, vitamin complexes and functional foods. In addition, in thepresent disclosure, the food may include special nutritional foods(e.g., milk formulas, baby food), meat products, fish meat products,tofu, jelled food, noodles (e.g., ramen, noodles), breads, dietarysupplements, seasonings food (e.g., soy sauce, soybean paste, red pepperpaste, and mixed soy paste), sauces, confectionery (e.g., snacks),candy, chocolate, gum, ice cream, milk products (e.g., fermented milk,cheese, etc.), other processed food, kimchi, pickled foods (variouskinds of kimchi, pickled vegetables, etc.), beverages (e.g., fruitdrinks, vegetable beverages, soy bean milk, fermented beverages, etc.)and natural seasonings (e.g., ramen soup), but are not limited thereto.The food, beverage or food additive may be prepared by a conventionalpreparation method.

In addition, the “functional food” means a food group imparted withadded value to function or express the function of the correspondingfood to a specific purpose by using physical, biochemical orbiotechnological techniques in food, or to control the bio-defenserhythm of the food composition, or food which is designed and processedso as to sufficiently express the body's control function regarding thebody, such as prevention of diseases and recovery, and the like, andspecifically, it may be a health functional food. The functional foodmay include a food-acceptable food-aid additive, and may further includesuitable carriers, excipients and diluents conventionally used in thepreparation of functional foods.

In the present disclosure, the term “beverage” means a generic term forquenching thirst or for enjoying a taste, and includes a functionalbeverage. The beverage includes a composition for preventing orameliorating the metabolic disease symptoms as an essential ingredientat the indicated ratio, and there is no particular limitation on theother ingredients. Various flavors or natural carbohydrates, such asordinary beverages, may be contained as additional ingredients.

In addition to those described above, the food for preventing orameliorating metabolic disease symptoms or containing the compositionfor fat oxidation of the present disclosure may contain variousnutrients, vitamins, minerals (electrolytes), flavors such as syntheticflavors and natural flavors, coloring agents and fillers (cheese,chocolate, etc.), pectic acids and its salts, alginic acids and itssalts, organic acids, protective colloid thickeners, pH adjustingagents, stabilizers, preservatives, glycerin, alcohol, and carbonationagents used for carbonated drink, and these ingredients may be usedindependently or in combination.

In the food containing the composition for preventing or amelioratingthe metabolic disease symptoms of the present disclosure or for fatoxidation, the amount of the composition according to the presentdisclosure may be in the range of 0.001% by weight to 90% by weight,based on the total weight of the whole food, and may include preferably0.1 to 40% by weight. In the case of beverages, they may be included inthe ratio of 0.001 to 2 g, preferably 0.01 to 0.1 g, based on 100 ml.However, for health and hygiene purposes, a long-term intake for thepurpose of health adjustment, the amount may be less than the aboverange. Since the active ingredient has no problem in terms of safety, itcan be used in an amount exceeding the above range, but is not limitedto the above range.

In addition, another aspect of the present disclosure provides atransformant expressing a lean phenotype, in which an Atg7 gene isdeleted in dendritic cells. In addition, another aspect of the presentdisclosure provides a method for producing a transformant expressing alean phenotype, in which the method includes deleting an Atg7 gene indendritic cells.

In addition, another aspect of the present disclosure provides apharmaceutical composition for preventing or treating metabolicdiseases, in which the pharmaceutical composition includes Atg7, and anexpression inhibitor or an activity inhibitor of a gene coding the sameas active ingredients.

The expression inhibitor of the gene coding the Atg7 may be thegene-specific siRNA (small interference RNA), shRNA (short hairpin RNA),miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), andantisense oligonucleotides.

The activity inhibitor of Atg7 may be an antibody, an aptamer, a naturalextract and chemicals specific for Atg7. The antibody may be amonoclonal antibody or a polyclonal antibody.

The term “transformation” in the present disclosure means a phenomenonthat the genetic properties of an organism are changed by DNA given fromthe outside, that is, when DNA, which is a kind of nucleic acidextracted from cells of any system of an organism, is given to livingcells of another system, the DNA enters into the cells to change genetictraits.

In the present disclosure, the term “transformant” means an individualproduced by transformation, and is not limited to the individual, butpreferably means a transformed animal.

In addition, another aspect of the present disclosure may provide a foodcomposition for preventing or ameliorating metabolic diseases, in whichthe food composition includes Atg7, an expression inhibitor or anactivity inhibitor of a gene coding the same as active ingredients.

In addition, one aspect of the present disclosure provides a method forscreening useful intestinal microorganisms, in which the method includescomparing intestinal microorganisms of an individual of obesity orgeneral phenotype with the transformant. The screening method mayfurther include, after the comparing, selecting (determining) amicroorganism having a large number of individuals in the transformantas useful intestinal microorganisms, as compared with intestinalmicroorganisms of an obese or general phenotype individual.

The useful intestinal microorganism may be a microorganism useful formetabolic diseases or fat oxidation, preferably a microorganism usefulfor lipid metabolic diseases.

Advantageous Effects

Bacteroides acidifaciens (BA) according to the present disclosureresults in the activation of fat oxidation through the bileacid-TGR5-PPARα axis in adipose tissue, resulting in high energyconsumption. At the same time, BA activates visceral DPP-4, followed bythe increase in GLP-1, thereby contributing to glucose homeostasis. Bileacids, cholate and taurine also contribute to GLP-1 activity through aTGR5 receptor and improve insulin sensitivity. Accordingly, it can beused as a very effective therapeutic agent or prevention agent ofmetabolic diseases.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing a lean phenotype in an Atg7^(ΔCD11c) mouse.

(A) The body weight change of Atg7^(f/f) and Atg7^(ΔCD11c) mice weremonitored for 23 weeks (left panel). Body weight and fat mass (rightpanel) of 24-week-old male Atg7^(f/f) and Atg7^(ΔCD11c) mice on a normalchaw diet (NCD). (n=8).

(B) Photographs of 24-week-old Atg7^(f/f) and Atg7^(ΔCD11c) mice.

(C) Mill of abdominal adipose tissue of 24-week-old male Atg7^(f/f) andAtg7^(ΔCD11c) mice on a NCD.

(D) Histological changes (left panel) and adipocyte size (right panel)in adipose tissue of Atg7^(f/f) and Atg7^(ΔCD11c) mice. Scale bars=50μm.

(E) Levels of glucose and insulin in the serum of Atg7^(f/f) andAtg7^(ΔCD11c) mice on a NCD under non-fasting conditions.

(F) GTT (Glucose tolerance test) and ITT (insulin tolerance test)results for male Atg7^(f/f) and Atg7^(ΔCD11c) mice.

All data are shown as mean±s.e.m. *P<0.05, **P<0.01, and ***P<0.001.

FIG. 2 is a diagram showing that a lean phenotype is originated fromvisceral symbiotic bacteria.

(A) Photographs of co-housing (CH; middle) and separated (left and rightends) 24-week-old male Atg7^(f/f) and Atg7^(ΔCD11c) mice.

(B) Body weight and fat mass of 24-week-old male Atg7^(f/f) andAtg7^(ΔCD11c) mice in a co-housing and a separated cage. (n=3 or 4).

(C) Results of monitoring the body weight of each mouse after co-housing(CH) for an additional 10 weeks (n=3 to 9).

(D) Body weight and fat mass of untreated B6 mice after 18 weeks ofmovement of feces in an Atg7^(f/f) or Atg7^(ΔCD11c) mouse (n=5).

(E) Levels of glucose and insulin in the serum after movement of fecalextract of Atg7^(f/f) or Atg7^(ΔCD11c) mouse under non-fastingconditions (n=5).

All data are mean±s.e.m values. *P<0.05, **P<0.01, ***P<0.001; ns, notsignificant.

FIG. 3 is a diagram showing that B. acidifaciens (BA) is expanded in thefeces of an Atg7^(ΔCD11c) mouse in visceral symbiotic bacteria.

(A) Phylum level and (B) pyrosequencing data (n=6) from the class to thegenus.

(C) Representative pie graphs of species representing the distributionof BA in the feces detected by pyrosequencing. Red arrow=BA.

(D) IECs (intestinal epithelial cells) of Atg7^(f/f) and Atg7^(ΔCD11c)mice determined by BA-specific FISH (fluorescence in situ hybridization)probe and the number of BA increased in lumens of colon (n=3). Scalebar=100 μm. All data are mean±s.e.m values. *P<0.05; ns, notsignificant.

FIG. 4 is a diagram showing that B. acidifaciens (BA) regulates the bodyweight and fat mass of diet-induced B6 mouse obesity.

(A) Photographs of high-fat diet (HFD; left panel) and PBS- and BA-fedmice. The body weight of each group was monitored for 10 weeks (rightpanel). BA was orally administered (5×10⁹ CFU/100 μl) (n=5).

(B) Oral dietary intake with PBS or BA (n=5).

(C) MRI of PBS- and BA-fed mice.

(D) Histological changes in adipose tissue (left panel) and adipocytesize (right panel) of PBS- and BA-fed mice in HFD.

All data are shown as mean±s.e.m of 2 independent experiments.

(E) Results of GTT (glucose tolerance test; left panel, n=8 or 9) andITT (insulin tolerance test; right panel, n=7-12) using the serum ofPBS- and BA-fed mice measured at a certain point after intraperitonealinjection of glucose or insulin.

(F) Energy consumption, total activity, and RER (respiratory exchangeratio) (n=5) of PBS- or BA-fed mice. All data are expressed asmean±s.e.m. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

FIG. 5 is a diagram showing that B. acidifaciens(BA) induces fatoxidation in adipose tissue through PPARα activity.

At the end of each experiment, the expression levels of mRNAs of genesrelated to fatty acid synthesis (FasN, HSL, PEPCK, SCD1, and PPARγ),β-oxidation (PPARα), thermogenesis (PRDM16, PGC1a, Cidea, and GLUT4)were determined through real-time PCR using the epididymis adiposetissues of Atg7^(f/f) and Atg7^(ΔCD11c) mice (A; n=5), mice transplantedwith fecal microorganisms (B; n=5), and BA-fed mice (C; n=5).

The PPARα (D) and TGR5 (E) expression level in adipose tissue after 1, 7and 14 days of daily BA administration were analyzed through RT-PCR.

All data are mean±s.e.m of 2 independent experiments. *P<0.05, **P<0.01,***P<0.001; ns, not significant.

FIG. 6 is a diagram showing that B. acidifaciens (BA) regulates DPP-4(dipeptidal peptidase-4) secretion to induce GLP-1 production.

(A) Levels of glucose and insulin in the serum of PBS- and BA-fed miceand (B) activated GLP-1 (B) (normal chow diet, NCD; high-fat diet, HFD;n=5).

(C) One hour after administration of BA or BA culture supernatant ormedium alone to untreated B6 mice, the level of DPP-4 in the smallintestine was confirmed by light emission analysis.

(D) Quantification of cholates and taurine in the feces of PBS- andBA-fed mice (n=5) using CE-MS (Capillary Electrophoresis MassSpectrometry).

All data are mean±s.e.m of 2 independent experiments. *P<0.05, **P<0.01,***P<0.001; ns, not significant.

FIG. 7 is a diagram showing a mechanism by which B. acidifaciens (BA)can prevent or treat insulin sensitivity and obesity.

Specific visceral symbiotic bacteria (i.e., BA) expanded in a leanphenotype Atg7^(ΔCD11c) mouse were identified. Administration of BAresults in the activation of fat oxidation through the bileacid-TGR5-PPARα axis in adipose tissue, resulting in high energyconsumption. At the same time, BA activates visceral DPP-4, followed bythe increase in GLP-1, thereby contributing to glucose homeostasis. Bileacids, cholate and taurine also contribute to GLP-1 activity through aTGR5 receptor and improve insulin sensitivity.

PPARα, peroxisome proliferator-activated receptor α; SCFAs, short-chainfatty acids.

FIG. 8 is a diagram showing that an Atg7^(ΔCD11c) mouse shows a leanphenotype regardless of gender.

Body weight of male (left) and female (right) Atg7^(f/f) andAtg7^(ΔCD11c) mice between 7 and 23 weeks of age on a NCD.

FIG. 9 is a diagram showing that the lean phenotype of 24-week-oldAtg7^(ΔCD11c) mouse is not associated with inflammation.

(A) Levels of pre-inflammatory cytokines in the serum of Atg7^(f/f) andAtg7^(ΔCD11c) mice (n=7) measured using a cytometric bead arraymouse-inflammatory kit (BD Biosciences).

(B) F4/80 (left) and TNFα (right) mRNA expression levels throughreal-time PCR.

(C) Results of hematoxylin eosin staining of small intestine and colon.Scale bar=100 μm.

All data are mean±s.e.m in 2 independent experiments. *P<0.05; ns, notsignificant.

FIG. 10 is a diagram showing OPLS-DA (Orthogonal partial least squaresdiscriminate analysis) of fecal metabolites of Atg7^(f/f) andAtg7^(ΔCD11c) mice.

(A) Cross-validated score plots from OPLS-DA of 1H-NMR (nuclear magneticresonance) in the feces of Atg7^(f/f) and Atg7^(ΔCD11c) mice (n=7).

(B) S-plot for the predicted components from OPLS-DA of 1H-NMR data inthe feces of Atg7^(f/f) and Atg7^(ΔCD11c) mice (n=7).

(C) Quantification of short chain fatty acids in the feces of Atg7^(f/f)and Atg7^(ΔCD11c) mice (n=5) using gas chromatography-mass spectrometry(e.g., acetates, butyrates and propinate) and lactate. All data aremean±s.e.m. *P<0.05, **P<0.01.

FIG. 11 is a diagram showing compensated body weight changes of a mouseof the same kind in co-housing (CH) cage and fecal microorganisms.

The body weight of Atg7^(f/f) (n=5) and Atg7^(ΔCD11c) (n=4) mice in a CHcage and the body weight of Atg7^(f/f) and Atg7^(ΔCD11c) mice (S) raisedseparately are shown by a line graph based on gray and blue circles,respectively.

FIG. 12 is a diagram showing that pyrosequencing data at the specieslevel in the feces of an Atg7^(ΔCD11c) mouse shown in FIG. 3C show acolor legend.

FIG. 13 is a diagram showing the biological arrangement of Bacteroidescontig in the DNA metagenome.

The relative abundance of the number of contigs in the feces ofAtg7^(f/f) and Atg7^(ΔCD11c) mice are compared. All data are shown asmean±s.e.m. *P<0.05; ***P<0.001; ns, not significant.

FIG. 14 is a diagram showing that B. acidifaciens (BA) that are orallyadministered remains in the colon temporarily.

Colons and feces were obtained at nil and after 1-5 days after oraladministration of BA (5×10⁹ CFU/100 μl), and stained with BA-specificFISH (fluorescence in situ hybridization) probes.

(A) The confocal image of the BA (yellow arrow).

(B) Quantification of BA in the feces at a specific point of time. Thenumber was counted at ≧20 sites per slide. All data are shown asmean±s.e.m of three independent experiments. ***P<0.001; N.D., notdetected.

FIG. 15 is a diagram showing the effective function of the body weightand fat mass of B. acidifaciens (BA) in NCD-fed B6 mice provided with BAor PBS.

(A) Photographs and body weight after 10 weeks (left and right panels,respectively). BA was orally administered daily (5×10⁹ CFU/100 μl).

(B) Oral dietary intake with PBS or BA.

(C) Mill analysis.

(D) Histological changes of adipose tissues (left panel) and changes inadipocyte size (right panel).

(E) Results of GTT (glucose tolerance test) (left panel, n=8 or 9) andITT (insulin tolerance test) (right panel, n=7-12) at a specific pointof time after intraperitoneal injection of glucose or insulin.

(F) Energy consumption, total activity, and RER (respiratory exchangeratio) of PBS- or BA-fed mice (n=5).

All data are shown as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001; ns, notsignificant.

FIG. 16 is a diagram showing that mice fed with NCD or HFD and B.sartorii (BS) have similar body weight and dietary intake.

(A) Body weight for 10 weeks after oral administration of BS (n=5)(5×10⁹ CFU/100 μl).

(B) Dietary intake.

Data are mean±s.e.m of two independent experiments. ns, not significant.

FIG. 17 is a diagram showing that the administration of B. acidifaciens(BA) improves liver and peripheral insulin sensitivity.

It is the result of performing hyperinsulinemic-euglycemic clamp forBA-, heat-inactivated BA-fed mice and NCD-fed control mice (n=3) for 6weeks.

During the clamp experiment, the amount of insulin solution wasdetermined to be 3 mU based on the first experiment. The inhibition ofsystemic glucose uptake (peripheral insulin sensitivity, A) andinsulin-mediated liver glucose production (liver insulin sensitivity, B)were significantly increased in BA-fed mice as compared to heatinactivated BA-fed group. All data are shown as mean±s.e.m. *P<0.05; ns,not significant.

FIG. 18 is a diagram showing the levels of SCFAs (short-chain fattyacids) and lactate levels in the feces after oral administration of B.acidifaciens (BA) for 10 weeks. Levels of acetate, butyrate, propionate,and lactate in the feces of mice with NCD (A; n=5) and HFD (B; n=6) weremeasured through gas chromatography-mass spectrometry.

All data are mean±s.e.m. *P<0.01; ns, not significant.

FIG. 19 is a diagram showing the similar levels of lipid metabolism inliver and small intestine in Atg7^(f/f) and Atg7^(ΔCD11c) mice.

The expression levels of mRNAs of genes related to fatty acid synthesis(FasN, HSL, PEPCK, SCD1, and PPARγ), β-oxidation (PPARα), thermogenesis(PRDM16, PGC1a, Cidea, and GLUT4) were determined through real-time PCRusing liver (A) and small intestine (B) of Atg7^(f/f) and Atg7^(ΔCD11c)mice (A; n=5).

All data are mean±s.e.m.

FIG. 20 is a diagram showing that B. acidifaciens (BA) does not induceβ-cell hyperpolarization.

Pancreatic tissues were obtained from mice (n=5) administered with(5×10⁹ CFU/100 μl) for 10 weeks.

(A) The confocal image of the pancreatic islet (α-cells are red, β-cellsare green). Scale bar=50 μm. Sections were continuously reacted withmouse anti-glucagon IgG Ab (K79bB10; Sigma-Aldrich, St. Louis, Mo.) andrabbit polyclonal anti-insulin Ab (Santa Cruz Biotechnology, Santa Cruz,Calif.), they were reacted with PE-conjugated anti-mouse IgG(eBioscience, San Diego, Calif.) and FITC-conjugated anti-rabbit IgG(eBioscience, San Diego, Calif.), respectively.

(B) The size of a β-cell region was quantified using an ImageJ softwareprogram. The pancreatic islets were randomly selected as 10 sites perslide. All data are mean±s.e.m. ***P<0.001; ns, not significant.

FIG. 21 is a graph showing triglyceride and cholesterol levels in plasmaof Atg7^(ΔCD11c), fecal microbiota transplantation (FMT), and B.acidifaciens (BA)-fed mice.

The concentrations of plasma triglycerides and total cholesterol wereanalyzed by using an enzyme assay kit in Atg7^(ΔCD11c) mice (A; n=3),FMT mice (B; n=5), and BA-fed mouse (C; n=5). All data are mean±s.e.m.ns, not significant. NCD, normal chow diet; HFD, high-fat diet.

FIG. 22 is a diagram showing that B. acidifaciens (BA) can regulateself-digestion of CD11c⁺ cells.

The number of BA in bone marrow-derived CD11c⁺ cells after 6 and 24hours of co-incubation with BA were determined on EG agar plates(MOI=10). Bone marrow was obtained from Atg7^(f/f) and Atg7^(ΔCD11c)mice. All data are mean±s.e.m of three independent experiments. *P<0.05;N.D., not detected.

Modes of the Invention

Hereinafter, the present disclosure will be described in more detail byway of examples. However, these examples are for illustrative purposesonly, and the scope of the present disclosure is not limited to theseexamples.

Example 1: Animal Experiment

All animal experiments were approved by the Asan Animal ExperimentalEthics Committee (permit number: PN 2014-13-069). All experiments wereperformed under anesthesia with ketamine (100 mg/kg) and xylazine (20mg/kg).

Example 2: Mice and Bacteria Strain

C57BL/6 (B6), and CD11c-Cre, Villine-Cre, and LysM-Cre mice werepurchased from Charles River Laboratories (Orient Bio Inc., Sungnam,Korea) and Jackson Laboratory (Bar Harbor, Me.). ATG7^(flox/flox) micewere provided by Masaaki Komatsu (Tokyo Metropolitan Institute ofMedical Science, Japan). Atg7^(ΔCD11c) mice were prepared bycrossbreeding CD11c^(cre) mice and ATG7^(f/f) mice in the animallaboratory of Seoul Asan Medical Center. All mice were fed with sterilefeed and drinking water under non-pathogenic conditions. B. acififaciens(JCM10556) and B. sartorii (JCM17136) were purchased from JapanCollection of Microorganisms (JCM) of RIKEN BioResource Center.

Example 3: 454 Pyrosequencing Analysis

cDNA was extracted from the feces using QIAamp DNA stool mini kits(Qiagen, Valencia, Calif.). PCR amplification was performed usingprimers targeting the V1 to V3 sites of 16S rRNA gene. For theamplification of bacteria, primer 9F(5′-CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-AC-AGAGTTTGATCMTGGCTCAG-3′; theunderlined sequence means primer at the target site) to which a barcodeis attached and 541R(5′-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC-ATTACCGCGGCTGCTGG-3′; ‘X’ meansa specific barcode of each object)(http://oklbb.ezbiocloud.net/content/1001) were used.

Amplification was performed under the following conditions: initiationof denaturation at 95° C. for 5 minutes, 30 cycles denaturation at 95°C. for 30 seconds, primer annealing at 55° C. for 30 seconds, andamplification at 72° C. for 30 seconds, and final extension at 72° C.for 5 minutes. The same concentrations of the presumed products werepooled together and short pieces (non-target objects) were removed usingan AMPure bead kit (Agencourt Bioscience, Beverly, Mass.). The qualityand size of the products were measured on Bioanalyzer 2100 (Agilent,Palo Alto, Calif.) using DNA 75001 chip. Sequencing of the mixedamplifications was performed via emulsion PCR, and then was placed on apicotiter plate. Sequencing was performed on Chunlab (Seoul, Korea) onthe GS Junior Sequencing System (Roche, Branford, Conn.). Pyrosequencingdata analysis was performed according to the prior art (Lim Y. W. etal.).

Example 4: Measurement of CE-TOF-MS (Capillary Electrophoresis (CE)Time-of-Flight Mass Spectrometry)

Quantitative analysis of the charged metabolites using CE-TOF-MS wasperformed as follows. 10 mg of lyophilized fecal samples were milledusing 3-mm zirconia-silica beads (BioSpec Products, Bartlesville,Okla.), and as an internal standard, they were homogenized using 400 μlof MeOH containing 20 μM each of methionine sulfone (Wako, Osaka, Japan)as a cation, IVIES (Dojindo, Kumamoto, Japan) as an anion, and CSA(D-Camphol-10-sulfonic acid; Wako). Subsequently, 200 μl of de-ionizedwater and 500 μl of chloroform were added. By using a Shakemaster neo(Bio Medical Science, Tokyo, Japan), they were stirred in 1,500 r.p.m.for 10 minutes, the solution was centrifuged at 4,600 g for 15 minutesat 4° C., and the protein was removed by filtering using a Millipore5,000-Da cut-off filter (Millipore, Billerica, Mass.). The filtrate waslyophilized and dissolved in 25 μl water containing 200 μM each of3-aminopyrrolidine (Sigma-Aldrich) and trimesate (Wako) as referencecompounds. All CE-TOF-MS experiments were conducted using AgilentTechnologies equipment: CE capillary electrophoresis system, G3250AALC/MSD TOF system, 1100 series binary HPLC pump, G1603A CE-MS adapter,and G1607A CE-ESI-MS sprayer kit. Data were treated (MasterHands) usinginternal software (Sugimoto M et al.) to determine peak annotation andquantification.

Example 5: Gas Chromatography Mass Spectrometry (GC-MS) Measurement

The organic concentration in the feces was determined by gaschromatography-mass spectrometry. A partial sample (80 ml) of the etherextract of the feces was mixed withN-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide. The vial wassealed, heated in boiling water at 80° C. for 20 minutes, and thenplaced at room temperature for 48 hours for derivatization. Thederivatized samples were treated with a 6890N Network GC System (AgilentTechnologies) equipped with an HP-5MS column (0.25 mm×30 m×0.25 mm) anda 5973 Network Mass Selective Detector (Agilent Technologies).

Pure helium (99.9999%) was used as carrier gas and was delivered at arate of 1.2 ml min⁻¹.

The outlet pressure was set at 97 kPa divided by 20:1. The inlet andtravel line temperatures were 250 and 260° C., respectively. Thetemperature program was used as follows: 60° C. (3 minutes), 60-120° C.(5° C./min), 120-300° C. (20° C./min). Subsequently, 1 μl of each samplewas injected for a reaction time of 30 minutes. The organic acidconcentration was quantified by comparing the peak area with thestandard.

Example 6: Measurement of GLP-1 (Glucagon-Like Peptide-1)

Blood samples were obtained from a control group and BA-fed mice, andwere centrifuged at 1800 g at 4° C. for 30 minutes. DPP-4 (dipeptidylpeptidase-4) inhibitor was added and GLP-1 concentration was determinedusing GLP-1 ELISA kit (Shibayagi).

Example 7: Measurement of DPP-4

DPP-4 levels were measured. After 6 hours of fasting in wild-type B6mice, BA (5×10⁹ CFU/100 μl) or its culture supernatant (100 μl/head) orculture medium alone was administered with DPP-4 inhibitor sitagliptin(40 mg/mouse; Merck Sharp Dohme and Chibret Laboratories, Rahway, N.J.)followed by glucose for 30 minutes. After 15 minutes, intestinalepithelial cells of the ileum were recovered from pretreated mice andwashed with PBS to remove luminal materials. Mucus was scraped off,epithelium was cut into 1-2 mm in length, and placed in 1 ml PBS. Thesliced tissues was spun down to centrifugation (6,000 g, 4° C., 5 min),and 50 μl of supernatant was incubated using DPP-4 Glo protease assay(Promega, Madison, Wis.) at 37° C. for 2 hours together with kitreagents. DPP-4 activity was calculated as the value of a control samplein the absence of sitagliptin.

Example 8: Statistics

GraphPad Prism software (GraphPad, La Jolla, Calif.) was used forstatistical analysis. Significant differences between the two groupswere analyzed by two-tailed paired t-test or Mann-Whitneyt t-test. Aplurality of groups were analyzed using two-way ANOVA followed byBonferroni post-hoc test (*, P<0.05; **, P<0.01; ***, P<0.001).

Example 9: Identification of Reduced Body Weight and Fat Mass inAtg7^(ΔCD11c) Mice

In order to identify the role of immune cell self-digestion action inthe occurrence of metabolic diseases, the body weight and action wereobserved in dendritic cells (Atg7^(ΔCD11c)), alimentary canal epithelialcells (Atg7^(Δvillin)), and macrophages (Atg7ΔLysM) of Atg7 conditionalknockout mice. The mice were fed with NCD (normal chow diet).Thereafter, it was confirmed that the difference in body weight betweenthe Atg7^(ΔCD11c) mice and their litter control group mice(Atg7^(flox/flox (f/f))) was increased (FIG. 1A).

Importantly, it was confirmed that 24-week-old Atg7^(ΔCD11c) mice hadvery low body weight and fat mass as compared to Atg7^(f/f) mice (FIGS.1, A and B). A lean phenotype (Atg7^(ΔCD11c); FIG. 8) is shown both infemales and males.

In MRI (Magnetic Resonance Imaging) analysis, abdominal adipose tissuesthat were remarkably reduced in Atg7^(ΔCD11c) mice were confirmed inboth axial and coronal directions as compared to their litter Atg7^(f/f)mice (FIG. 1C). In addition, the single adipocyte size of visceraladipose tissue obtained from Atg7^(ACD11c) mice was significantly smallas compared to Atg7^(f/f) mice (FIG. 1D).

In order to confirm the involvement of systemic or mucosal inflammationin Atg7^(ΔCD11c) mice of a lean phenotype, the level of proinflammatorycytokine in serum and mRNA expression of F4/80 and TNFα in visceraladipose tissue were confirmed, and the tissues of the small intestineand colon were analyzed. It was confirmed that Atg7^(ΔCD11c) mice showedsimilar or decreased levels of multiple markers of systemic and mucosalinflammation indicating that the lean phenotype of Atg7^(ΔCD11c) micewas not related to inflammation (FIG. 9).

Importantly, higher insulin and subsequent low glucose levels thanAtg7^(f/f) mice under non-fasting conditions were identified in theserum of Atg7^(ΔCD11c) mice (FIG. 1E). It was confirmed that the insulinresistance determined by GTT (glucose tolerance test) and ITT (insulintolerance test) in Atg7^(ΔCD11c) mice as compared to the litterAtg7^(f/f) was improved in Atg7^(ΔCD11c) mice as compared to the litterAtg7^(f/f) (FIG. 1F). To sum up, these data indicate that Atg7^(ΔCD11c)mice have reduced fat mass and improved glucose homeostasis.

Example 10: Identification of Low SCFAs Levels in the Feces ofAtg7^(ΔCD11c) Mice

Since aged Atg7^(ΔCD11c) mice have low body weight and fat mass, therelevance between a lean phenotype and energy use was confirmed usingCE-TOF-MS (capillary electrophoresis time-of-flight mass spectrometry)of Example 4 and GC-MS (gas chromatography mass spectrometry) of Example5 in the feces.

Individual plots of Atg7^(ΔCD11c) mice in OPLS-DA (orthogonal partialleast squares discriminant analysis) are clearly distinct fromAtg7^(f/f) mice (FIG. 10A).

Furthermore, some SCFAs such as acetate, butyrate, propionate andlactate are located at the remote spot from the axis (FIG. 10B), whichindicates that these factors contribute to distinguish Atg7^(f/f) andAtg7^(ΔCD11c) mice. In reality, the amount of acetate, butyrate, andpropionate in Atg7^(ΔCD11c) mice was remarkably reduced as compared toAtg7^(f/f) mice, whereas the amount of lactate was higher (FIG. 10C).

Example 11: Identification that Symbiotic Bacteria are Related to a LeanPhenotype of Aged Atg7^(ΔCD11c) Mice

In order to confirm whether a lean phenotype of Atg7^(ΔCD11c) mice isrelated to symbiotic bacteria, co-housing (CH) and FMT (fecal microbiotatransplantation) experiments were performed. From birth, Atg7^(ΔCD11c)and Atg7^(f/f) mice shared a cage and exposed feces.

As a result, Atg7^(f/f) mice that shared the cage with Atg7^(ΔCD11c)lost more both body weight and fat as compared to Atg7^(f/f) mice (FIGS.2, A and B; FIG. 11). In addition, Atg7^(ΔCD11c) mice that shared thecage with Atg7^(f/f) mice increased body weight and fat compared toAtg7^(ΔCD11c) mice (FIGS. 2, A, and B; FIG. 11). In order to confirmwhether the phenotype of CH mice was due to symbiotic microorganisms,mice were transferred to their respective cages after co-housingexperiments. As shown in FIG. 2C, Atg7^(ΔCD11c) mice lost body weightwhen they were raised alone, whereas Atg7^(f/f) mice did not. Inaddition, the oral administration of the fecal extract of Atg7^(ΔCD11c)mice to wild-type B6 mice for 12 weeks resulted in significantly lowerbody weight and fat mass than the feces of wild type B6 or Atg7^(f/f)mice (FIG. 2D). In addition, importantly, wild-type B6 mice to which thefecal extract of Atg7^(ΔCD11c) mice is administered showed higherinsulin levels and subsequent low serum glucose levels as compared tothe mice to which the extract of Atg7^(f/f) is administered (FIG. 2E).

To sum up, these results indicate that they play an essential role inthe lean phenotype of Atg7^(ΔCD11c) mice.

Example 12: Expansion of Bacteroides acidifaciens (BA) in the Feces ofAtg7^(ΔCD11c) Mice

Metagenomics analysis was used to confirm the diversity and compositionof intestinal symbiotic bacteria. In pyrosequencing analysis, it wasconfirmed that the faces of Atg7^(f/f) and Atg7^(ΔCD11c) mice weremutually similar in the primary distribution of intestinalmicroorganisms at the Bacteroidetes, Firmicutes, and Proteobacteriaratios, and phylum level (FIG. 3A). There was no similar or significantdifference in the distribution of Bacteroidia (class), Bacteroidales(order), Bacteroidaceae (family), and Bacteroides (genus) (FIG. 3B).However, at the species level, it was confirmed that the ratio of BA wassignificantly extended in the feces of Atg7^(ΔCD11c) mice as compared toAtg7^(f/f) mice (5.48±1.76% vs. 0.77±0.18%) (FIG. 3C, red arrow; FIGS.12 and 13).

On the other hand, there was no difference in the ratio of the otherBacteroides species including B. sartorii in the feces of Atg7^(ΔCD11c)or Atg7^(f/f) mice (FIG. 13).

In alpha diversity, the species abundance of the fecal microorganisms ofAtg7^(ΔCD11c) mice (Chao 1 index) was remarkably reduced, whereas thebiodiversity (Shannon/Simpson index) was similar to the fecalmicroorganism of Atg7^(f/f) mice (Table 1 below).

TABLE Simpson Chao1 Shannon Reciprocal Atg7^(f/f) 923.13 ± 109.72 4.39 ±0.43 0.04 ± 0.02 Atg7^(ΔCD11c)  604.41 ± 203.83* 4.28 ± 0.28 0.04 ± 0.03

FISH (fluorescence in situ hybridization) analysis was performed toconfirm the expansion of BA in Atg7^(ΔCD11c) mice of a lean phenotype.As shown in FIG. 3D, the number of increased BA was detected in thelumen of the colon, and it was confirmed that a small amount of BA wasinternalized into the colon epithelial cells of Atg7^(ΔCD11c) mice.

To sum up, these results indicate that, in symbiotic bacteria, BA hasbeen expanded in lean phenotype intestines.

Example 13: Identification that the Oral Administration of BA to HighFat Diet (HFD)-Provided B6 Mice Induces a Lean Phenotype

In order to confirm whether extended BA regulates lipid metabolism, BA(JCM10556) was obtained and cultured to obtain large amounts ofmicroorganisms, which were provided to untreated B6 mice.

In order to determine the optimal conditions of administration, colontissues and BA in mice fed with BA (5×10⁹CFU/100 μl) were quantitated byFISH analysis. One day after oral administration, a large number of BAwere detected in the lumen of colon epithelial cells (FIG. 14A).

After 2 days of oral administration, the number of BA in the peak fecessubsequently disappeared and recovered rapidly (FIG. 14B). It wasconfirmed that the oral administration of BA reduced body weight and fatmass of wild-type B6 mice provided with NCD and HFD without dietaryeffects (FIG. 4, A-C; FIG. 15, A-C). In contrast, no body weight losswas observed in B. sartorii-provided mice used as control groups (FIG.16). In addition, the size of single adipocytes in adipose tissue of theepididymal was significantly lower in BA- and HFD-provided B6 mice ascompared to PBS- and HFD-provided mice (FIG. 4D). In addition, theinsulin resistance determined by GTT and ITT improved remarkably in BA-and HFD-provided mice as compared to PBS- and HFD-provided mice (FIG.4E).

A hyperinsulinemic-euglycemic clamp technique using heat-inactivated BAas a control group was used to confirm the effect of BA feeding on liverand peripheral insulin sensitivity.

Interestingly, the BA feeding improved liver and peripheral insulinsensitivity (FIG. 17). It was confirmed that the oral administration ofBA showed a decrease in butylate in the feces of NCD-fed mice, but itwas confirmed that levels of acetate, propionate, and lactate were notchanged (FIG. 18A). A similar trend was confirmed in the HFD-fed group(FIG. 18B). In order to measure energy consumption, activity andsubstrate utilization, the mice provided with BA were monitored afterthey were individually raised in a CLAMS (a comprehensive laboratoryanimal monitoring system) cage for 5 days. It was confirmed that thegroup of PBS or BA-provided mice showed a similar locomotor activity andrespiratory exchange ratio, whereas BA- and HFD-fed mice consumed moreenergy as compared to PBS- and HFD-fed mice (FIG. 4F). The NCD-fed miceto which oral BA is administered showed a similar effect (FIG. 15).

To sum up, the long-term administration of BA induces energyconsumption, and accordingly, causes a lean phenotype of dominance indiet-induced obese mice.

Example 14: Identification that a Lean Phenotype Mouse Shows an Increasein PPARα (Peroxisomeproliferator-Activated Receptor α) Expression inAdipose Tissues

Expression levels of genes related to lipid metabolism in adiposetissue, liver, and small intestine were analyzed based on the detectionof decreased body weight and fat mass in Atg7^(ΔCD11c) FMT B6, andBA-fed B6 mice. Importantly the expression of genes related to lipidβ-oxidation, particularly PPARα, increased only in adipose tissue of theepididymis of Atg7^(ΔCD11c) mice (FIG. 5A). No significant differencecould be identified in the small intestine and liver (FIGS. 19A and B).To be consistent with these results, PPARα expression was significantlyup-regulated in adipose tissue of the epididymis of B6 mice fed with thefecal extract of Atg7^(ΔCD11c) and mice fed with HFD and BA for 10 weeks(FIGS. 5, B and C).

The level of PPARα expression in B6 mice by BA administration by thetime-dependent method was measured to confirm whether the enhancedβ-oxidation level was activated by the bacteria alone or was notactivated by the product of a lean phenotype.

Interestingly, the level of mRNA of PPARα in epididymis adipose tissueof B6 mice was significantly increased after 2 weeks of BAadministration (FIG. 5D).

In addition, the expression levels of TGR5, GPBAR1 (G-protein-coupledbile acid receptor), which can stimulate energy consumption throughPPARα activity, were measured.

As a result, it was confirmed that BA administration increased the levelof TGR5 expression in adipose tissue (FIG. 5E). These results indicatethat a lean phenotype by BA initiates fat oxidation in adipose tissueaccording to TGR5-PPARα activity.

Example 15: Identification that BA Mediates Production of GLP-1(Glucagon-Like Peptide-1) by Regulation of DPP4 (Dipeptidylpeptidase-4)and Production of Bile Acids

The role of BA in glucose homeostasis was confirmed. As expected, BA-fedB6 mice showed higher insulin and lower glucose levels in serum thanPBS-fed B6 mice (FIG. 6A).

In order to confirm whether this increase in plasma insulin levels wasdue to over-stimulation of β-cells, α and β cells were stained inpancreatic tissue 10 weeks after BA feeding.

As a result, it was confirmed that the BA feeding did not inducehypersensitivity of β-cells (FIG. 20).

In order to confirm the mechanism of high levels of insulin secretion inBA-fed lean mice, levels of GLP-1 stimulating insulin release into theblood were measured.

GLP-1 levels in serum were remarkably increased in NCD and HFD-fed mice(FIG. 6B).

It was confirmed that the level of DPP-4, a well-known enzyme withinhibiting activity of GLP-1, decreased in the small intestine and ileumafter oral administration of BA or its culture supernatant (FIG. 6C).

In addition, DPP-4 activity was measured, reflecting the amount ofprotein. Previous studies have shown that bile juice plays a key role inglucose homeostasis through stimulation of GLP-1 secretion through TGR5activity.

As a result, it was confirmed a significantly increased level ofdeconjugated cholate, salt of cholic acid, and taurine from primary bileacid in the feces of B6 mice fed with BA for 10 weeks, but significantloss of cholesterol could not be confirmed (FIG. 6D; FIG. 21). Theseresults indicate that BA or its metabolites lower the activity of theDPP-4 enzyme, and accordingly, causing GLP-1 activity, thereby improvinginsulin sensitivity and glucose resistance.

To sum up, the present inventors have confirmed that the specificintestinal symbiotic bacteria (i.e., BA) are extended in the leanphenotype Atg7^(ΔCD11c) mice. The administration of BA results in theactivation of fat oxidation through the bile acid-TGR5-PPARα axis inadipose tissue, resulting in high energy consumption.

At the same time, BA activates visceral DPP-4, followed by the increasein GLP-1, thereby contributing to glucose homeostasis. Bile acids,cholate and taurine contributed to GLP-1 activity through a TGR5receptor and improved insulin sensitivity.

Accordingly, it can be understood that BA plays an important role in theprevention or treatment of metabolic diseases such as diabetes andobesity (FIG. 7).

1. A pharmaceutical composition for preventing or treating metabolicdiseases, the pharmaceutical composition comprising Bacteroidesacidifaciens as an active ingredient.
 2. The pharmaceutical compositionfor preventing or treating metabolic diseases according to claim 1,wherein the metabolic diseases are one or more types selected from thegroup consisting of obesity, diabetes, diabetic complication, fattyliver, hypertension, peripheral vascular disease, dyslipidemia, insulinresistance, cardiovascular disease, arteriosclerosis, metabolicsyndrome, hyperglycemia, hyperlipidemia, and carbohydrate metabolicabnormality.
 3. The pharmaceutical composition for preventing ortreating metabolic diseases according to claim 1, wherein theBacteroides acidifaciens has a higher ratio of intestinal total bacteriain a lean phenotype as compared to obesity or a standard phenotype. 4.The pharmaceutical composition for preventing or treating metabolicdiseases according to claim 1, wherein the Bacteroides acidifaciensactivates fat oxidation in adipose tissue, inhibits the activity ofintestinal DPP-4 (dipeptidal peptidase-4), and increases GLP-1.
 5. Acomposition for oxidizing fat, the composition comprising Bacteroidesacidifaciens as an active ingredient.
 6. A composition for inhibitingDPP-4 (dipeptidal peptidase-4), the composition comprising Bacteroidesacidifaciens as an active ingredient.
 7. A food composition forimproving or preventing metabolic diseases, the food compositioncomprising Bacteroides acidifaciens as an active ingredient.
 8. Atransformant expressing a lean phenotype, wherein an Atg7 gene isdeleted in dendritic cells.
 9. A method for producing a transformantexpressing a lean phenotype, comprising: deleting an Atg7 gene indendritic cells of an individual.
 10. A pharmaceutical composition forpreventing or treating metabolic diseases, the pharmaceuticalcomposition comprising Atg7, and an expression inhibitor or an activityinhibitor of a gene coding the same as active ingredients.
 11. A methodfor preventing or treating metabolic diseases, the method comprisingadministering Bacteroides acidifaciens to a subject in need ofprevention or treatment of metabolic diseases.
 12. A use of Bacteroidesacidifaciens for preventing or treating metabolic diseases.
 13. A use ofBacteroides acidifaciens to be used for the preparation of apharmaceutical composition for preventing or treating metabolicdiseases.
 14. A method for oxidizing fat or inhibiting DPP-4, the methodcomprising administering Bacteroides acidifaciens to a subject in needof fat oxidization or DPP-4 inhibition.
 15. A use of Bacteroidesacidifaciens for oxidizing fat or inhibiting DPP-4.
 16. A use ofBacteroides acidifaciens to be used for the preparation of apharmaceutical composition for oxidizing fat or inhibiting DPP-4.